- Email: [email protected]
[88] Citrate-cleavage enzyme
[88]
CITRATErCLEAVAGE ENZYME
[ 88]
641
Citrate-Cleavage Enzyme
Mg++ Citrate + ATP + coenzyme A
--* Acetyl coenzymo A + oxalacetate + ADP + P~
B y PATJL A. SRERE General. This enzyme has been referred to by two other names in the literature. It was first called the citrate ATP CoA enzyme1 and was later referred to as the citrate-activating enzyme.2 Since recent evidence indicates that the reaction probably occurs toward citrate breakdown, the present name has been employed. A partial purification of this enzyme from pigeon liver has been published. 1 This present procedure has been worked out for use with chicken liver2
Assay Method Principle. When 'citrate is incubated with ATP, CoA, Mg ~, and enzyme, two of the products formed are acetyl CoA and OAA. The enzyme can be assayed by trapping the acetyl CoA as acetylhydroxamate and measuring the latter by the color produced with FeCla,1 but this assay is useful only for a small range of enzyme concentration. A larger range of enzyme concentrations can be assayed when 0AA is measured by its reaction with D P N H in the presence of malic dehydrogenase. Reagents
0.1 M Tris buffer, pH 7.3. 0.1 M MgC12. 0.2 M potassium citrate. CoA, 10 mg./ml. (Pabst). 0.1 M mereaptoethanol. DPNH, 2.5 mg./ml. Malic dehydrogenase~ 10,000 units/ml. 0.1 M ATP. Procedure. The following materials are added to a 1.5-ml. silica cell (length = 1 cm.): 0.1 ml. of Tris, 0.1 ml. of MgCl2, 0.1 ml. of
l p. A. Srere and F. Lipmann, J. Am. Chem. Soc. 75, 4874 (1953). " M. Wolleraan, Ac~a Physiol. Acad. Sci. Hung. 10, 171 (1956). P. A. Srere, J. Biol. Chem. $$4, 2544 (1959).
642
ENZYMES OF CITRIC ACID CYCLE
[88]
potassium citrate, 0.1 ml. of mercaptoethanol, 0.05 ml. of malic dehydrogenase, 0.03 ml. of coenzyme A, 0.05 ml. of D P N H , enzyme to be assayed, and water to make a total volume of 0.95 ml. A blank cell is prepared with all components except D P N H and CoA. The reaction is started by adding 0.05 ml. of ATP. The decrease in absorption at 340 m~ is measured. The rate is usually linear, and any time period is acceptable for rate determination. This assay is applicable to crude tissue extracts as prepared by the method described. Other extraction methods usually yield extracts with considerable D P N H oxidase, which must be removed for accurate assays. The oxidation of D P N H is proportional to enzyme activity in the range 0.001 to 0.03 unit of activity. Protein Determination. Protein concentration is measured by a modification of the turbidimetrie method of Biieher. 4 Definition o] Unit. One unit of activity is equal to 1 micromole of D P N H oxidized per minute at 20 ° in the assay described. Purification Procedure
Extraction. Fresh or frozen chicken liver (45 g.) is homogenized in a Waring blendor with 450 ml. of 0.4 M KCI in 2 0 % ethanol. The initialtemperature of the KCl-ethanol solution is 0 °, but homogenization is allowed to proceed for 2 minutes at room temperature. Subsequent operations are carried out with the enzyme solution in a beaker in an ice bath. Centrifugations are also performed at 0% After homogenization, the extract is allowed to stand for 5 minutes, and the fatty layer that rises to the top is skimmed from the extract. The homogenate is centrifuged at 20,000 X g for 15 minutes. The supernatant fluid is poured through several layers of cheesecloth. The filtrate is referred to as the enzyme extract. I. Acidification. For each liter of extract 60 ml. of 1 M sodium acetate buffer, p H 5.4, is added, and the solution is stirred for 15 minutes, centrifuged at 20,000 X g for 15 minutes, and the precipitate discarded. This step invariably shows an increase in total activity over the original extract. 2. Zinc Step. To each liter of supernatant fluid, I0 ml. of 0.1 M zinc acetate is added. The solution is stirred for 15 to 30 minutes and centrifuged at 20,000 X g for 15 minutes. The supernatant fluid is discarded, and the precipitate is suspended in cold 0.5 M KCI (one-twentieth the volume of the originalextract) by means of a glass homogenizer. The suspension is centrifuged at 20,000 X g for 10 minutes, and the precipitate is re-extracted with 0.5 M KCI in a similar manner. Occasionally T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
[88]
CITRATE-CLEAVAGE ENZYME
643
ethylenediaminetetraacetate (0.1 M) has been used to help dissolve the precipitate. 3. Alcohol Step. The combined KC1 solutions (protein concentrations between 22 and 26 mg./ml.) are diluted with cold water to a KC1 concentration of 0.1 M. The solution is stirred for 15 minutes, and the precipitate is removed by centrifugation. To the supernatant fluid (protein concentration between 3 and 6 mg./ml.) cold alcohol is added to a final concentration of 15%. The precipitate is collected by centrifugation and dissolved in water or dilute KC1. In some instances this quantity of alcohol is added in steps to give two to four different precipitates. Precipitates formed in passing from 7 to 12% alcohol contain the btflk of the activity. 4. Ammonium Sul/ate Step. The enzyme preparation obtained by alcohol fractionation is treated with ammonium sulfate, either in saturated solution or as the crystalline salt. The protein concentration of the alcohol fraction was usually 20 mg./ml., although fractionation has been carried out at both higher and lower protein concentrations. The increment in ammonium sulfate concentration precipitating the bulk of the activity is variable depending on the solvent that has been used for dissolving the alcohol precipitate. If 0.5M KC1 has been used, the precipitate forming between 25 and 33% saturation with ammonium sulfate contains most of the activity, but if water has been used as the solvent, the activity falls in the fraction appearing between 30 and 40% saturation. 5. Alumina C~ Gel Step. The enzyme solution is adjusted to pH 5.9 with a 1 M sodium acetate buffer, pH 5.4, and added to a quantity of alumina C~ gel (previously packed by eentrifugation) such that there is 1 mg. of gel for each milligram of protein. The solution is allowed to stand for 15 minutes at 0 °. The gel is collected by centrifugation, and the supernatant fluid is discarded. The gel is washed with 0.04 M potassium citrate, pH 7.0, this wash being discarded. The enzyme is recovered by washing the gel with 0.1 M potassium citrate, pH 7.0. Notes on Purification. A study of the influence of various extraction media on the completeness of extraction has been made. It has been found that 0.4 M KC1 in 20% ethanol gives extracts of maximum activity. Special precautions in preparing the original extract, such as adding the alcohol slowly at 0 ° to --5 °, do not affect the activity obtained. Chicken liver frozen for as long as 3 months retains most of its activity. The table shows the results of fifteen separate purification trials carried through the ammonium sulfate step. The success with alumina C~ gel has been variable. The highest specific activities obtained are in the range of 1.4 to 1.6, or an over-all purification of a hundredfold.
644
ENZYMES OF CITRIC ACID CYCLE
[88]
In each fractionation step except the gel step a complete recovery of all activity can be made. A number of other procedures is effective in concentrating enzyme activity, but no evidence for separation into several proteins has been obtained, even when such procedures as chromatogSUMMARY OF PURIFICATION OF CITRATE-CLEAVAGE ]~NZYME
(Purification was usually performed on 90 or 135 g. of chicken liver. The numbers in parentheses indicate the range'of activities for fifteen separate purification trials. The other figures are average values for the fifteen runs.) Step Enzyme extract Acidification Zinc Alcohol Ammonium sulfate
Specific activity, X 102 Total activity/g. Recovery 1.0 (0.72-1.64) 2.0 (1.15-2.52) 10.1 (4.45-15.5) 24 (11.1-51.0) 59.5 (22.1-127)
1.5 1.8 1.3 0.91 0.53
100 123 87 60 35
raphy on diethylaminoethyl cellulose or on calcium phosphate gel columns have been used. Whenever any procedure causes a significant loss of activity, recombination of fractions is tried, but stimulation of one fraction by any or all of the others has never been observed. Properties
Distribution. The enzyme is found to be widely distributed in animal tissues. The greatest enzyme activities are observed in extracts of chicken liver and rat liver. I t is present in rat, rabbit, pig, chicken, and beef tissues (kidney, heart, brain, liver). Extracts of spinach and of mushroom have no enzyme activity. Stability. All the fractions are unstable. A variety of conditions and additives have no effect on the stability of the enzyme. Typically, 40% of the activity is lost on storage of the enzyme overnight. Dialysis against distilled water for 4 hours does not affect the activity. The enzyme can be diluted to protein concentrations of less than 0.1 mg./ml. without apparent loss of activity within 15 minutes. Heating at 48 ° for 1 minute destroys 90% or more of the enzyme activity. Enzymatic Purity. The enzyme after gel purification contains little or no condensing enzyme activity, no D P N H oxidase, no malic dehydrogenase, no acetyl CoA deacylase, traces of ATPase, and traces of myokinase.
CITRATErCLEAVAGE ENZYME
[ 88]
641
Citrate-Cleavage Enzyme
Mg++ Citrate + ATP + coenzyme A
--* Acetyl coenzymo A + oxalacetate + ADP + P~
B y PATJL A. SRERE General. This enzyme has been referred to by two other names in the literature. It was first called the citrate ATP CoA enzyme1 and was later referred to as the citrate-activating enzyme.2 Since recent evidence indicates that the reaction probably occurs toward citrate breakdown, the present name has been employed. A partial purification of this enzyme from pigeon liver has been published. 1 This present procedure has been worked out for use with chicken liver2
Assay Method Principle. When 'citrate is incubated with ATP, CoA, Mg ~, and enzyme, two of the products formed are acetyl CoA and OAA. The enzyme can be assayed by trapping the acetyl CoA as acetylhydroxamate and measuring the latter by the color produced with FeCla,1 but this assay is useful only for a small range of enzyme concentration. A larger range of enzyme concentrations can be assayed when 0AA is measured by its reaction with D P N H in the presence of malic dehydrogenase. Reagents
0.1 M Tris buffer, pH 7.3. 0.1 M MgC12. 0.2 M potassium citrate. CoA, 10 mg./ml. (Pabst). 0.1 M mereaptoethanol. DPNH, 2.5 mg./ml. Malic dehydrogenase~ 10,000 units/ml. 0.1 M ATP. Procedure. The following materials are added to a 1.5-ml. silica cell (length = 1 cm.): 0.1 ml. of Tris, 0.1 ml. of MgCl2, 0.1 ml. of
l p. A. Srere and F. Lipmann, J. Am. Chem. Soc. 75, 4874 (1953). " M. Wolleraan, Ac~a Physiol. Acad. Sci. Hung. 10, 171 (1956). P. A. Srere, J. Biol. Chem. $$4, 2544 (1959).
642
ENZYMES OF CITRIC ACID CYCLE
[88]
potassium citrate, 0.1 ml. of mercaptoethanol, 0.05 ml. of malic dehydrogenase, 0.03 ml. of coenzyme A, 0.05 ml. of D P N H , enzyme to be assayed, and water to make a total volume of 0.95 ml. A blank cell is prepared with all components except D P N H and CoA. The reaction is started by adding 0.05 ml. of ATP. The decrease in absorption at 340 m~ is measured. The rate is usually linear, and any time period is acceptable for rate determination. This assay is applicable to crude tissue extracts as prepared by the method described. Other extraction methods usually yield extracts with considerable D P N H oxidase, which must be removed for accurate assays. The oxidation of D P N H is proportional to enzyme activity in the range 0.001 to 0.03 unit of activity. Protein Determination. Protein concentration is measured by a modification of the turbidimetrie method of Biieher. 4 Definition o] Unit. One unit of activity is equal to 1 micromole of D P N H oxidized per minute at 20 ° in the assay described. Purification Procedure
Extraction. Fresh or frozen chicken liver (45 g.) is homogenized in a Waring blendor with 450 ml. of 0.4 M KCI in 2 0 % ethanol. The initialtemperature of the KCl-ethanol solution is 0 °, but homogenization is allowed to proceed for 2 minutes at room temperature. Subsequent operations are carried out with the enzyme solution in a beaker in an ice bath. Centrifugations are also performed at 0% After homogenization, the extract is allowed to stand for 5 minutes, and the fatty layer that rises to the top is skimmed from the extract. The homogenate is centrifuged at 20,000 X g for 15 minutes. The supernatant fluid is poured through several layers of cheesecloth. The filtrate is referred to as the enzyme extract. I. Acidification. For each liter of extract 60 ml. of 1 M sodium acetate buffer, p H 5.4, is added, and the solution is stirred for 15 minutes, centrifuged at 20,000 X g for 15 minutes, and the precipitate discarded. This step invariably shows an increase in total activity over the original extract. 2. Zinc Step. To each liter of supernatant fluid, I0 ml. of 0.1 M zinc acetate is added. The solution is stirred for 15 to 30 minutes and centrifuged at 20,000 X g for 15 minutes. The supernatant fluid is discarded, and the precipitate is suspended in cold 0.5 M KCI (one-twentieth the volume of the originalextract) by means of a glass homogenizer. The suspension is centrifuged at 20,000 X g for 10 minutes, and the precipitate is re-extracted with 0.5 M KCI in a similar manner. Occasionally T. Biicher, Biochim. et Biophys. Acta 1, 292 (1947).
[88]
CITRATE-CLEAVAGE ENZYME
643
ethylenediaminetetraacetate (0.1 M) has been used to help dissolve the precipitate. 3. Alcohol Step. The combined KC1 solutions (protein concentrations between 22 and 26 mg./ml.) are diluted with cold water to a KC1 concentration of 0.1 M. The solution is stirred for 15 minutes, and the precipitate is removed by centrifugation. To the supernatant fluid (protein concentration between 3 and 6 mg./ml.) cold alcohol is added to a final concentration of 15%. The precipitate is collected by centrifugation and dissolved in water or dilute KC1. In some instances this quantity of alcohol is added in steps to give two to four different precipitates. Precipitates formed in passing from 7 to 12% alcohol contain the btflk of the activity. 4. Ammonium Sul/ate Step. The enzyme preparation obtained by alcohol fractionation is treated with ammonium sulfate, either in saturated solution or as the crystalline salt. The protein concentration of the alcohol fraction was usually 20 mg./ml., although fractionation has been carried out at both higher and lower protein concentrations. The increment in ammonium sulfate concentration precipitating the bulk of the activity is variable depending on the solvent that has been used for dissolving the alcohol precipitate. If 0.5M KC1 has been used, the precipitate forming between 25 and 33% saturation with ammonium sulfate contains most of the activity, but if water has been used as the solvent, the activity falls in the fraction appearing between 30 and 40% saturation. 5. Alumina C~ Gel Step. The enzyme solution is adjusted to pH 5.9 with a 1 M sodium acetate buffer, pH 5.4, and added to a quantity of alumina C~ gel (previously packed by eentrifugation) such that there is 1 mg. of gel for each milligram of protein. The solution is allowed to stand for 15 minutes at 0 °. The gel is collected by centrifugation, and the supernatant fluid is discarded. The gel is washed with 0.04 M potassium citrate, pH 7.0, this wash being discarded. The enzyme is recovered by washing the gel with 0.1 M potassium citrate, pH 7.0. Notes on Purification. A study of the influence of various extraction media on the completeness of extraction has been made. It has been found that 0.4 M KC1 in 20% ethanol gives extracts of maximum activity. Special precautions in preparing the original extract, such as adding the alcohol slowly at 0 ° to --5 °, do not affect the activity obtained. Chicken liver frozen for as long as 3 months retains most of its activity. The table shows the results of fifteen separate purification trials carried through the ammonium sulfate step. The success with alumina C~ gel has been variable. The highest specific activities obtained are in the range of 1.4 to 1.6, or an over-all purification of a hundredfold.
644
ENZYMES OF CITRIC ACID CYCLE
[88]
In each fractionation step except the gel step a complete recovery of all activity can be made. A number of other procedures is effective in concentrating enzyme activity, but no evidence for separation into several proteins has been obtained, even when such procedures as chromatogSUMMARY OF PURIFICATION OF CITRATE-CLEAVAGE ]~NZYME
(Purification was usually performed on 90 or 135 g. of chicken liver. The numbers in parentheses indicate the range'of activities for fifteen separate purification trials. The other figures are average values for the fifteen runs.) Step Enzyme extract Acidification Zinc Alcohol Ammonium sulfate
Specific activity, X 102 Total activity/g. Recovery 1.0 (0.72-1.64) 2.0 (1.15-2.52) 10.1 (4.45-15.5) 24 (11.1-51.0) 59.5 (22.1-127)
1.5 1.8 1.3 0.91 0.53
100 123 87 60 35
raphy on diethylaminoethyl cellulose or on calcium phosphate gel columns have been used. Whenever any procedure causes a significant loss of activity, recombination of fractions is tried, but stimulation of one fraction by any or all of the others has never been observed. Properties
Distribution. The enzyme is found to be widely distributed in animal tissues. The greatest enzyme activities are observed in extracts of chicken liver and rat liver. I t is present in rat, rabbit, pig, chicken, and beef tissues (kidney, heart, brain, liver). Extracts of spinach and of mushroom have no enzyme activity. Stability. All the fractions are unstable. A variety of conditions and additives have no effect on the stability of the enzyme. Typically, 40% of the activity is lost on storage of the enzyme overnight. Dialysis against distilled water for 4 hours does not affect the activity. The enzyme can be diluted to protein concentrations of less than 0.1 mg./ml. without apparent loss of activity within 15 minutes. Heating at 48 ° for 1 minute destroys 90% or more of the enzyme activity. Enzymatic Purity. The enzyme after gel purification contains little or no condensing enzyme activity, no D P N H oxidase, no malic dehydrogenase, no acetyl CoA deacylase, traces of ATPase, and traces of myokinase.